Emerging evidence implicating ubiquitination in the pathogenesis of cancer has renewed interest in designed therapies that manipulate protein ubiquitination. Reversible ubiquitin modification of target proteins on lysine residues results in major changes in signaling proteins, either through degradation through the proteasome, or by altering the activity, localization, or protein complexes. Dysregulation of protein ubiquitination has been linked to cancer thus raising the promise that better understanding of these signaling proteins can be combined with chemistry to create new therapeutics. A number of key pathways have been shown to be regulated by protein ubiquitination. Based on these observations, there is increased enthusiasm for targeting proteins affecting protein ubiquitination. Strategies include targeting components of the E1-E2-E3 cascade or targeting deubiquitinating enzymes (DUB) that affect disease processes. A number of studies have identified potential DUB inhibitors (DUBi) as potential targets. Chemical or genetic loss of function studies targeting USP7, USP14, USP9X, and UCHL5 have been reported with anti-tumor properties. This project is meant to begin studies nominating protein ubiquitination targets by focusing on developing chemical biology strategies to profile DUB activity in cancer cells and tumor tissues, and define mechanism of action of early DUB inhibitor compounds. We propose to use a chemical biology platform, activity-based protein profiling (ABPP), to study the activity of DUB in lung cancer and nominate new potential targets. ABPP uses chemical probes that are directed against the active sites of enzymes to interrogate the functional state of enzymes in biological samples. These activity-based probes have two critical elements: (i) a group that reacts with enzymes and covalently labels their active sites and (ii) a reporter tag that allows for the detection or enrichment of the modified enzymes for detection using MS approaches. Preliminary data with DUB ABPP probes demonstrates feasibility of using this technology to profile DUB and the effects of DUBi in lung cancer cells. Aim 1 will characterize DUB proteins in lung cancer cell lines and tumors using ABPP. DUB probes will be used to characterize DUB signaling in multiple histological subtypes of lung cancer, including adenocarcinoma, squamous cell, and small cell. Tumor tissues taken at the time of surgical resection and established cell lines will be profiled and annotated to established DUB signaling pathways. Aim 2 will interrogate the functional significance of discovered DUB proteins identified using ABPP to identify important targets that mediate lung cancer viability. The first approach is a candidate approach, where DUB identified using ABPP will be examined for their role in promoting lung cancer growth and survival using RNA interference and small molecule inhibitors in cell line models. The second approach is to identify ?driver? DUB using a target-agnostic phenotypic screen of DUBi with target identification using ABPP. Changes in cell viability induced by DUBi in lung cancer cell lines will be used alongside competitive DUB profiling to nominate & validate targets.